Method and apparatus for monitoring a condition in chlorophyll containing matter

ABSTRACT

A method of monitoring health in chlorophyll containing matter comprises exposing the matter to a light source to cause chlorophyll to fluoresce and emit a fluorescence signal. Any changes in a parameter indicative of changes in the intensity of the fluorescence signal are detected and compared with a predetermined threshold. A change which exceeds the predetermined threshold is interpreted as a transition of the level of stress in the chlorophyll containing matter. An apparatus for monitoring health in chlorophyll containing matter is also provided and comprises a light source for causing chlorophyll in the matter to fluoresce, a detector for detecting the intensity the fluorescent signal, means for measuring changes in a parameter indicative of changes in the intensity of the fluorescent signal and a detector to detect an increase in the change of the parameter above a predetermined threshold.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/CA01/01039, filed Jul. 16, 2001, which was published in English,and which claims priority from U.S. Provisional Patent Application No.60/218,141, filed Jul. 14, 2000 and Canadian Patent Application No.2,352,639, filed Jul. 6, 2001.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for monitoring acondition in chlorophyll containing matter, for example fruits,vegetables and plants. The present invention also relates to a methodand apparatus for controlling environmental conditions in which fruit,vegetables and plants can be stored over prolonged periods of time.

BACKGROUND OF THE INVENTION

A number of techniques presently exist for extending the time over whichfruit and vegetables can be successfully stored without seriouslyaffecting their quality between harvest and consumption. Such storagetechniques are used to preserve various crops during transportation fromone part of the world to another and to make seasonal commoditiesavailable to the consumer during other parts of the year.

Fresh fruits and vegetables are living tissues which continue to respireafter harvesting. The process of respiration involves the use of oxygenin breaking down the food reserve contained within the fruit orvegetable, releasing energy and producing carbon dioxide. The rate ofrespiration, and therefore the rate of loss of the food reserve anddeterioration of the commodity, is closely related to the respirationrate.

To prolong the storage periods of fruits and vegetables, theirrespiration rate is reduced by lowering the temperature and oxygenlevels of the environment in which they are stored and by allowing thecarbon dioxide level to increase. However, lowering the temperature toofar will cause damage by freezing or chilling injury. Reducing theoxygen concentration too much will cause fermentation to occur withinthe fruit or vegetable which accelerates the ageing process and possiblycauses other forms of damage associated with low oxygen levels. Astorage environment containing excessive concentrations of CO₂ can alsocause damage to fruit and vegetables. Damage resulting from incorrectenvironmental storage conditions reduces the quality and marketpotential of the produce.

The precise level of temperature, oxygen and carbon dioxide required tomaximize storage life and to minimize storage disorders varies widely,depending on the type of produce, cultivars, growing conditions,maturity, harvest conditions, and post-harvest treatments. The idealstorage conditions can also depend upon where the particular product isgrown and can vary from season to season. Recommended levels fordifferent kinds of produce, which may be based, for example, on a crop'sstorage behaviour in previous years, are published by various nationalresearch bodies and extension advisors, and are considered to be thebest compromise between extending life and minimizing storage disorders.The storage facilities are controlled to maintain the storageenvironment for a particular product at these recommended fixed levels.Because of the number of factors and their variability on which theideal storage conditions depend, maintaining the product at therecommended levels may result in premature damage, in which case storageof the product has to be curtailed or loss is incurred. On the otherhand, as the recommended levels often include a safety margin above aknown damage threshold, the respiration rate of the produce isnecessarily above the minimum the produce can tolerate, possibly leadingto a shortened storage time.

A system for controlling the air composition in a room for storingvegetable products is disclosed in International Patent Application,Publication No. WO-A-96/18306. In one example, the system includescarbon dioxide and oxygen sensors for sensing the carbon dioxide andoxygen content, respectively, of a storage room in which vegetableproducts are stored. Under the control of a computer processor, theoxygen level in the storage room is reduced and the ratio between thecarbon dioxide and oxygen levels is monitored. For normal respiration,the amount of carbon dioxide produced by the stored product isapproximately equal to the oxygen consumed by the product so that theratio of carbon dioxide to oxygen should be and remain equal toapproximately 1, as the oxygen level is reduced. If the oxygen level isdecreased too far, fermentation occurs where no oxygen is consumed butcarbon dioxide is still produced, in which case the ratio of carbondioxide to oxygen becomes greater than 1. The control system reduces theoxygen content until the latter condition is observed and thereafterincreases the oxygen content slightly. If the ratio returns to 1, theoxygen content is again lowered until an increase in the ratio isdetected. In another example, the occurrence of fermentation in thestored vegetable product is detected directly by measuring the presenceof metabolites such as ethanol or lactate, formed by the fermentationprocess. In this case, the oxygen content is lowered until the presenceof ethanol or lactate in the storage room is detected by a sensor andthereafter the oxygen content is slightly increased. If the increase issufficient to bring the ethanol or lactate levels down to anunmeasurable level, the oxygen content is again gradually decreaseduntil a measurable amount of lactate or ethanol is detected.

A method of testing the post-harvest quality of fruits and vegetables,such as firmness, texture, aroma and color using chlorophyllfluorescence is disclosed in U.S. Pat. No. 5,822,068. The methodinvolves irradiating a fruit or vegetable sample firstly with low levelred light to stimulate minimal fluorescence within the chlorophyll anddetecting the intensity of the minimal fluorescence, Fo, emitted by thesample, and shortly thereafter irradiating the sample with high levelred light to stimulate maximum fluorescence within the chlorophyll anddetecting the maximal fluorescence intensity, Fm, emitted by the sample.A relatively high value of either of these signals is taken as anindication of good quality, whereas lower values in the fluorescencesignals are correlated to lower quality in the product.

Chlorophyll fluorescence techniques have also been used to detect damageand disorders in apples caused by low oxygen levels. One such study isdescribed in: The Proceedings of the 7th Controlled AtmosphereConference, Volume 2, pp 57–64 (1997), “Chlorophyll fluorescence detectslow oxygen stress in “Elstar” apples”, R. K Prange, S. P. Schouten andO. van Kooten, in which the minimal fluorescence intensity signal Of andthe ratio (Fm−Fo)/Fm were measured for Elstar apples stored over aperiod of 20 days in an atmosphere containing 0.07% oxygen. The resultsshow that Fo increased over the test period whereas (Fm−Fo)/Fmdecreased. Independent quality measurements indicated that some of thelow oxygen treated samples were firmer than the control samples, whichwere stored in air, and that the only disorder observed in the lowoxygen treated apples was a gradual increase in an off-flavour duringthe 20 day treatment period.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided amethod of monitoring stress in chlorophyll containing matter, comprisingthe steps of exposing the matter to a light source to cause chlorophyllin the matter to fluoresce and emit a fluorescence signal, detecting anychanges in a parameter indicative of changes in the intensity of thefluorescence signal, comparing any changes with a predeterminedthreshold and interpreting a change which exceeds the predeterminedthreshold as a transition of the level of stress in the matter.

The inventors have found that the onset of stress in chlorophyllcontaining produce is detectable by measuring chlorophyll fluorescenceand is signified by an increase in the change of fluorescence intensity.

In one embodiment, the detected parameter is the intensity of thefluorescence signal.

According to another aspect of the present invention, there is providedan apparatus for monitoring stress in chlorophyll containing matter,comprising a light source for causing chlorophyll in the matter tofluoresce and emit a fluorescence signal, a detector for detecting theintensity of the fluorescence signal, means for measuring changes in aparameter indicative of changes in the intensity of the fluorescencesignal, and means arranged to detect an increase in the change of theparameter above a predetermined threshold.

According to another aspect of the present invention, there is provideda method of controlling the intensity of a light source for stimulatinga fluorescence signal from chlorophyll containing matter, comprising thesteps of pulsing the light source and controlling the intensity of thelight source by controlling the time period over which the light sourceis pulsed.

According to another aspect of the present invention, there is providedan apparatus for stimulating a fluorescence signal from chlorophyllcontaining matter comprising a light source, means for pulsing theintensity of the light source, and a controller for controlling the timeperiod over which the light source is pulsed.

According to another aspect of the present invention, there is providedan apparatus for detecting a fluorescence signal emitted fromchlorophyll-containing matter comprising a detector for detecting theintensity of the fluorescence signal, means for recording the intensityof each of a plurality of fluorescence signals over time, means forcomparing a parameter responsive to the intensity of the fluorescencesignal with a predetermined value and means for indicating when ameasured intensity exceeds the predetermined value.

According to another aspect of the present invention, there is provideda method of determining an optimum value of an environmental parameterof an environment for storing chlorophyll-containing fruit orvegetables, comprising the steps of: (a) exposing the fruit or vegetableto a light source to cause chlorophyll in the fruit or vegetable tofluoresce and emit a fluorescence signal (b) detecting the intensity ofthe fluorescence signal, (c) measuring the value of the changingenvironmental parameter, (d) progressively reducing the oxygen level,(e) measuring the change in the intensity of the fluorescence signal asthe environmental parameter is changed, (f) detecting an increase in thechange of the intensity of the fluorescence signal, and (g) determiningthe optimal value of the environmental parameter from the detectedincrease in the change of the fluorescence intensity.

According to another aspect of the present invention there is providedan apparatus for determining an optimum value of an environmentalparameter of an environment for storing chlorophyll containing fruit orvegetables comprising: a light source to cause chlorophyll in said fruitor vegetable to fluoresce and emit a fluorescence signal, a detector fordetecting the intensity of said fluorescence signal, a sensor formeasuring the value of an environmental parameter, control meansarranged to progressively change said environmental parameter, means formeasuring changes in the fluorescence intensity as the value of saidenvironmental parameter is progressively changed, and means arranged todetect an increase in the change of the fluorescence intensity above apredetermined threshold.

According to the present invention there is further provided a systemand method for controlling an environmental parameter in a storage roomfor storing chlorophyll containing fruit and vegetables in response tochanges in the intensity of chlorophyll fluorescence emitted by theproduce.

BRIEF DESCRIPTION OF THE DRAWINGS

Examples of embodiments of the present invention will now be describedwith reference to the drawings in which:

FIG. 1 shows a block diagram of an apparatus for measuring thefluorescence response of chlorophyll containing matter in accordancewith an embodiment of the present invention;

FIG. 2 shows a cross-sectional view of an apparatus for measuring thefluorescence response of chlorophyll containing matter in accordancewith an embodiment of the present invention;

FIG. 3 shows an example of a graph of fluorescence intensity as afunction of integral photon flux of a chlorophyll fluorescencestimulating light source;

FIGS. 4A, 4B and 4C show examples of light source pulsing methods inaccordance with embodiment of the present invention;

FIG. 5 shows an example of a graph of relative fluorescence intensity asa function of the integral photon flux emitted by a fluorescencestimulating light source;

FIGS. 6A to 6D show examples of measurements of low oxygen stress inapple samples;

FIGS. 7A to 7D show examples of measurements of low oxygen stress inavocado samples;

FIGS. 8A to 8D show examples of measurements of low oxygen stress inbanana samples;

FIGS. 9A to 9D show examples of measurements of low oxygen stress inkiwi fruit samples;

FIG. 10A to 10D show examples of measurements of low oxygen stress inmango samples;

FIG. 11A to 11D show examples of low oxygen stress in pear samples;

FIGS. 12A to 12D show examples of measurements of low oxygen stress incabbage samples;

FIGS. 13A to 13D show examples of measurements of low oxygen stress ingreen pepper samples;

FIGS. 14A to 14D show examples of measurements of low oxygen stress iniceberg lettuce samples;

FIGS. 15A to 15D show examples of measurements of low oxygen stress inromaine lettuce samples;

FIGS. 16A to 16E show examples of measurements of high carbon dioxidestress in cabbage samples;

FIGS. 17A to 17C show examples of measurements of low temperature stressin banana samples;

FIGS. 18A and 18B show examples of moisture stress in strawberry plants;

FIG. 19 shows a schematic diagram of a system for performing a methodaccording to an embodiment of the invention;

FIG. 20 shows a schematic diagram of the gas control system of FIG. 19;

FIG. 21 shows a diagram of the control valve arrangement of the gascontrol system of FIG. 20;

FIG. 22 shows a diagram of the gas analyzer system of FIG. 20;

FIG. 23 shows a top view of an embodiment of a fluorometer used in thesystem of FIG. 19;

FIG. 24 shows an arrangement of light sources and light sensors of thefluorometer shown in FIG. 23;

FIG. 25A shows a graph of the variation of chlorophyll fluorescence withoxygen concentration for an apple sample;

FIG. 25B shows a table of numerical data plotted in the graph of FIG.25A;

FIG. 26A shows a graph of the variation of chlorophyll fluorescence withoxygen concentration for a kiwi fruit sample;

FIG. 26B shows a table of part of the numerical data plotted in thegraph of FIG. 26A;

FIG. 27A shows a graph of the variation of chlorophyll fluorescence withoxygen concentration for a mango sample;

FIG. 27B shows a table of part of the numerical data plotted in thegraph of FIG. 27A;

FIG. 28A shows a graph of the variation of chlorophyll fluorescence withoxygen concentration for a pear sample;

FIG. 28B shows a table of part of the numerical data plotted in thegraph FIG. 28A;

FIG. 29A shows a graph of the variation of chlorophyll fluorescence withoxygen concentration for an avocado sample;

FIG. 29B shows a table of part of the numerical data plotted in thegraph of FIG. 29A;

FIG. 30A shows a graph of the variation of fluorescence with oxygenconcentration for a banana sample;

FIG. 30B shows a table of part of the numerical data plotted in thegraph of FIG. 30A;

FIG. 31 shows a table of the results of measured firmness in applesamples stored over a period of 4 months under two different conditions,and

FIGS. 32A and 32B shows a graph of Fα and FO measured during a simulatedNitrogen Flush accident.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows an embodiment of an apparatus for detecting stress inchlorophyll containing produce according to an embodiment of the presentinvention. The apparatus, generally shown at 1 comprises a rectangulararray of four light sources, 3, 5, 7, 9 and a light sensor 11 positionedwithin a central region of the rectangular array of light sources, allmounted on a support panel 13. In this embodiment, the light sources 3,5, 7, 9 each comprises a light emitting diode (LED) although in otherembodiments, any other suitable light source may be used. The sensorcomprises a photodiode, although in other embodiments, any othersuitable light sensor may be used. An optical filter 14 is arranged infront of the sensor 13 to prevent the sensor receiving light from thelight sources. The apparatus further includes a driver 15 for drivingthe light sources and a controller 17 which may for example be amicroprocessor, for controlling the light sources. The apparatus alsoincludes an amplifier 19 for amplifying signals from the light sensor 11and an analog to digital converter (ADC) 21 for converting the amplifiedanalog signal from the amplifier 19 into a digital signal. The output ofthe A/D converter is connected to an input of the controller 17. Acomputer 23 interfaces with the controller 17 to control the operationof the light sources and to record data relating to the signals detectedby the light sensor 11. The components of the apparatus enclosed withinthe dashed line 25 may optionally be housed within a single enclosure orcasing for convenience.

FIG. 2 shows an example of an arrangement in which the apparatus is usedto monitor stress in chlorophyll containing produce.

Referring to FIG. 2, one or more produce samples 103 are placed in acontainer 105 having a removable lid 107. A stress sensing device 101 ispositioned within the container 105 such that the light sources 103 andthe light sensor 111 are directed at the sample(s) 127. The stresssensing device 101 is positioned a fixed distance from the producesample(s) 127 and may conveniently be mounted to the inside of the lid107 of the container.

In the arrangement shown in FIG. 2, the distance between the stresssensing device 101 and the produce sample 127 is such that the lightsources combine to irradiate a relatively high proportion of theaccessible upper surface area of the sample and the sensor can receivefluorescence signals emitted from a relatively high proportion of thesample area.

An example of a method of monitoring stress in chlorophyll containingmatter will now be described with reference to FIGS. 1 and 2.

The computer 23 specifies a predetermined light source intensity levelto the controller 17 which causes the driver 15 to energize the lightsources 3, 5, 7 and 9 at the specified intensity level. The lightsources may be energized such that the light intensity at the samplesurface is generally at an intensity in the range of about 0.01 to 10μmol·m⁻²·s⁻¹, or more. A fluorescence signal emitted by the chlorophyllin response to light from the light sources is detected by the sensor 11and converted into an electrical signal which is amplified by theamplifier 19, converted to a digital signal by the A/D converter, andthe resulting digital signal is read by the controller 17 and passed tothe computer 23 for processing and storage.

In one embodiment, the computer calculates any change in the intensityof the fluorescence signal by comparing a value of intensity measured atone time with the value of the intensity at another time. The computermay be arranged to provide an indication to a user of the measuredfluorescence intensity so that the user can compare the current value ofintensity with previous values and thereby monitor any change. Such anindication may be provided visually, for example on visual display, orby any other suitable means. The computer 23 may also be arranged tocompare any change with a predetermined threshold value and provide anindication of when a change in the intensity of the fluorescence signalexceeds the predetermined threshold.

In a preferred embodiment, the controller 17 controls the A/D converter21 to process signals from the sensor both when the light source is onand when the light source is off. The measured intensities resultingfrom both conditions are then read by the microprocessor and passed tothe computer 23. The computer 23 subtracts the intensity measured whenthe light sources are off from the intensity measured when the lightsources are on to provide an intensity value which is solelyattributable to fluorescence stimulated by the light source without anycontribution from other possible background sources.

In one embodiment of a method of monitoring stress in chlorophyllcontaining produce, the fluorescence intensity is measured for aplurality of different amounts of light or light levels. Thismethodology may be implemented under the control of the computer whichmay be arranged to instruct the controller to energize the light sourceat a first level of integrated photon flux and shortly thereafter toenergize the light sources with a second, different level of integratedphoton flux. The computer receives the fluorescence intensity valuesmeasured at each light level and may record them in memory for furtherprocessing. In this way, the intensity of the fluorescence signalemitted from the chlorophyll may be measured for many different lightsource intensity levels.

The inventors have discovered that the fluorescence intensity emitted bychlorophyll as a function of the light source can be generally describedmathematically, for example by a second order polynomial.Advantageously, measuring the fluorescence intensity at a number ofdifferent light source levels allows the values of the parametersdescribing the polynomial to be calculated, one of which is the valueF_(α) which is the theoretical value of the fluorescence intensity whenthe light source intensity is zero. The inventors have discovered thatF_(α) can be measured with a high degree of accuracy and is extremelysensitive to physiological changes in the chlorophyll due to stress. Theparameters A and B which describe the second and first order terms ofthe polynomial, respectively, have also been discovered to be usefulindicators of the physiological state of the chlorophyll containingproduce.

An example of the dependence of fluorescence intensity on the lightsource intensity level is shown in FIG. 3. Data points are representedby crosses and the polynomial regression fitted to the data points isshown by the continuous line. The value of F_(α) corresponds to thevalue of fluorescence intensity at the intercept of the extrapolatedcurve at the Y axis (i.e. at light source level=zero).

A particularly advantageous method of varying the intensity of the lightsource to permit data to be taken at many different light source levelswill now be described with reference to FIGS. 4A, 4B and 4C.

Embodiments of the present invention vary the photon flux to which thechlorophyll containing matter is exposed by on-off pulsing the lightsource and varying various parameters which define the pulses. In onemethod of generating a given light level, a train or series of pulses isgenerated each having a defined pulse width and a predefined timebetween pulses. In this case, the effective light level is theintegrated photon flux over the train sequence. To generate a differenteffective light level, a second train of pulses is generated having thesame pulse width and number of pulses but a different time intervalbetween pulses. An example of this method is shown in FIG. 4A. FIG. 4Ashows two pulse trains both having the same number of pulses 405 (inthis case four although any suitable train of pulse could be used ineach train e.g. between 1 and 1000 or more) and with the pulses in bothpulse trains having the same pulse width, t₁. However, the time t₂between pulses is longer for the first pulse train 401 than the secondpulse train 403. Therefore, the integrated photon flux for the secondpulse train 403 is greater than the integrated photon flux of the firstpulse train 401.

In another embodiment, the light source level or integrated photon fluxis varied by varying the pulse width, an example of which is shown inFIG. 4B. FIG. 4B shows two pulse trains 409, 411, both pulse trainshaving the same number of pulses 413, 415. However, the pulse width t₃of the pulses 413 of the first pulse train 409 is less than the pulsewidth t₄ of the pulses 415 of the second pulse train 411. The pulseperiod t₅ is the same for both pulse trains and therefore the timebetween pulses t₆ for the first pulse train 409 is greater than the timet₇ between pulses of the second pulse train 411. Thus, the integratedphoton flux is larger for the second pulse train 411 than for the firstpulse train 409.

The pulsing technique exemplified in FIG. 4A may be referred to as pulsefrequency modulation (PFM) since the frequency of the pulses is variedbetween different pulse train sequences to vary the integrated photonflux. In this case, as the frequency is increased from 1/t₈ to 1/t₉, theintegrated photon flux changes from F1 to F2, where F2=F1×t9/t8. Thepulsing technique shown in FIG. 4B in which the pulse width is varied tochange the integrated photon flux may be referred to as pulse widthmodulation (PWM). In PWM mode, the pulse period t₅ remains constant andthe pulse width is varied. As the pulse width is increased from t₃ tot₄, the integrated photon flux changes from F3 to F4, where F4=F3×t₄/t₃.The controller is capable of generating either one or both forms ofpulse sequence. In the examples of fluorescence measurements describedbelow and shown in the drawings, the increase in integrated photon fluxis represented on the X axis as “LED duty cycle”, which for PFM modewould be t₁/t₈ (or t₁/t₉) and for PWM mode would be t₃/t₅ (or t₄/t₅).The LED duty cycles may typically vary between 0.00002 and 0.06, whichrepresents an integrated photon flux of approximately 0.01 μmol·m⁻²·S⁻¹to ¹⁰ μmol·m⁻²·S⁻¹.

Another example of a pulsing technique which may be used to vary theintegrated photon flux is shown in FIG. 4C. In this embodiment, theintegrated photon flux is varied by varying the time period of eachpulse train sequence.

Referring to FIG. 4C, the controller 17 controls the driver to pulse theintensity of the light sources at a predetermined frequency. For eachfluorescence intensity measurement, the light sources are pulsed over apredetermined period of time T₁, T₂. Thus, the intensity of light towhich the chlorophyll is exposed is the integral of the light intensityof each pulse over that time period. Thus, the intensity may be variedvery sensitively, i.e. in very small increments, simply by changing thewidth of the time period over which the light sources are pulsed.

After each time period, the light source may be turned off so that thebackground fluorescence can be measured and subtracted from the data.

In another embodiment, the integrated photon flux may be varied byvarying the intensity of the pulses. In other embodiments, theintegrated photon flux may be varied by varying a combination of any twoor more parameters which define a sequence of pulses.

The fluorescence received by the sensor may be measured when the lightsource is off, for example after each pulse in a pulse train, after someof the pulses, or at the end of each pulse train, or less frequently.The measured background fluorescence may be subtracted from the measuredfluorescence intensity during each pulse to provide a measurement of thefluorescence intensity emitted solely in response to the light source.

The monitoring apparatus may be generally arranged to sample thefluorescence response of the chlorophyll containing matter periodically,for example under the control of a computer 23 (shown in FIG. 1). In oneembodiment, a sample may involve exposing the chlorophyll containingmatter to a single level of photon flux and detecting the intensity ofthe fluorescence signal emitted in response thereto. For example, thechlorophyll containing matter may be exposed to light having anintensity which stimulates a relatively low level of fluorescence, forexample minimal fluorescence fo, or near minimal fluorescence, or ahigher level of actinic fluorescence.

In another embodiment, a sample may involve exposing the chlorophyllcontaining matter sequentially to a plurality of different levels ofintegrated photon flux and detecting the fluorescence intensity or aparameter indicative of the fluorescence intensity at each level ofphoton flux.

A level of photon flux may be generated by energizing the light sourceat a predetermined intensity continuously for a predetermined period oftime and the level of photon flux may be changed by energizing the lightsource at a different predetermined intensity continuously for asubsequent predetermined period of time. Between each photon flux level,the intensity of the light source may be reduced, for example to 0. Thefluorescence intensity may be measured in response to each differentlevel of photon flux and may also be measured when the light source isoff, for example between changes in the intensity of the light source,so that the background fluorescence can be subtracted from the measuredfluorescence at each different level of photon flux.

In other embodiments of the present invention, one or more differentlevels of photon flux may be generated by generating a predeterminedseries of light pulses. In this case, the level of photon flux is theintegrated photon flux over the series of pulses. The integrated photonflux of each series of pulses depends on the parameters which define thepulses in each series, such as pulse intensity, pulse width, pulse rate(or frequency) and the number of pulses in each series, for example asdescribed above in conjunction with FIGS. 4A to 4C. One or more of theseparameters may be defined by an operator and input into a control systemsuch as a computer by a user interface (e.g. graphical use interface(GUI), mouse, or keyboard). A plurality of different levels ofintegrated photon flux may be generated sequentially by generating aplurality of different series of pulses and by changing one or moreparameters defining each series, for example the parameters mentionedabove. For a single sample scan, the integrated photon flux may beprogressively increased or decreased or different integrated photonfluxes may be generated in any selected or arbitrary order. Preferably,each scan includes at least three different levels of integrated photonflux to allow a second order polynomial to be parameterized to the data.The accuracy to which the parameters describing the polynomialregression can be determined increases with the number of fluorescenceintensity data at different levels of integrated photon flux, andadvantageously, this technique of generating different levels ofintegrated photon flux by pulsing the light source allows the integratedphoton flux to be changed very precisely in very small increments,allowing a single scan to include many different levels of integrated ofphoton flux. For example, a single scan may include between 10 and 500or more different levels of photon flux and typical measurements havebeen made using about 200 different levels of integrated photon flux.Other parameters which define a pulse scan may include the start and endlevels of integrated photon flux, the increment of integrated photonflux between each successive level and the length of any pause (i.e.time period) between each successive level of integrated photon flux. Inone embodiment where the level of integrated photon flux isprogressively changed by changing the frequency (i.e. pulse rate)between successive series of pulses, parameters such as pulse width,start pulse rate, end pulse rate, pulse rate steps, number of pulses tobe repeated at each pulse rate and pause between pulse rate steps may beselected.

In one embodiment, the chlorophyll fluorescence intensity may bemeasured during each pulse of a series of pulses defining a level ofintegrated photon flux. A single fluorescence intensity may bedetermined for each level of integrated photon flux by calculating theaverage fluorescence intensity of some or all of the fluorescenceintensity measurements at each pulse.

Preferably, the fluorescence intensity is measured during each pulse andbetween each pulse (when the light source is off to provide anindication of the background fluorescence, e.g. due to any backgroundlight). The fluorescence intensity (D) after the light pulse is thensubtracted from the fluorescence intensity (L) during the light pulse toprovide a relative fluorescence (F), where F=L−D. Again, the averagerelative fluorescence intensity is determined over some or all of theseries of pulses as mentioned above. For each fluorescence intensitymeasurement at each level of integrated photon flux, the duty cycle ofthe light source is also calculated as P=PW/PP, where PW is the pulsewidth and PP is the pulse period. As shown in FIG. 5, the fluorescenceintensity generally increases with the level of integrated photon flux(which may be expressed as the light source duty cycle).

A second order polynomial regression of the form F=A×P²+B×P+Fα isparameterized to the F vs. P curve. The F vs. P curve shitis and changesshape as chlorophyll containing matter experiences stress, for exampleto changing environmental parameters and this can be observed in thecurve parameterizations A, B and Fα. Since a scan can typicallyrepresent a significant number of data points, these parameters havebeen found to be extremely stable when the chlorophyll containing matteris not undergoing physiological changes (e.g. due to stress). Asmentioned above, the parameter Fα represents the value of F that wouldbe measured at P=0 (a theoretical point at which the integrated photonflux approaches 0), which has been shown to be a proxy for thetraditional reading of Fo. The A and B parameters represent thecurvature and slope of the F vs. P curve. These two parameters are verysensitive to physiological changes in chlorophyll containing matter e.g.fruits, vegetables and plants. The parameters Fα, A and B may becalculated from the data for example by a computer program. Changes inthese parameters may also be calculated or may be observed by displayingthe data visually, for example on a computer screen. Changes in theseparameters may be compared with a determined level of change and theoccurrence of a change above a predetermined level may be detected and,for example, signified to an operator. The detection of the parameterexceeding a predetermined level of change may signify that the health ofthe chlorophyll containing matter is being stressed, and such adetermination may be used to control a parameter effecting thechlorophyll containing matter, for example an environmental parametersuch gaseous mixture, temperature, moisture level, pressure or any otherinfluence to which parameters derived from fluorescence intensitymeasurements are sensitive.

While a constant (i.e. non-scanning) pulse can be used to repeatedlymeasure a single fluorescence reading, it can be difficult to detectchanges from that reading because of inherent noise levels. Thus, thescanning technique is preferred because of its greater sensitivity tochange. Methods, e.g. algorithms which automatically signal significantchange, for example as defined by an operator, can be defined generallyor specifically for different applications.

Changes in the fluorescent measurements have been shown to directlyrelate to changes in the “health” of the chlorophyll containing mattercaused by variations, for example, in environmental conditions andparameters such gaseous mixture, temperature, time and moisture. Thus,the state of the health of chlorophyll containing matter (e.g. plants,fruit and vegetables) due to their environment, whether stable,improving or under stress, can be determined with this methodology sothat appropriate action can be taken. The detection of a significantchange in a parameter based on the chlorophyll fluorescence may be usedin an automated system as a control signal to control a parameteraffecting the health of the chlorophyll containing matter concerned.

Examples of measurements of the onset of stress and recovery from stressin the health of various chlorophyll containing matter such as fruitsand vegetables which are produced by changes in a variety of differentenvironmental conditions will be described below.

Monitoring Health in Fruit Varieties at Low Oxygen Concentrations

The following examples illustrate how embodiments of the presentinvention can be used to detect the onset of low oxygen stress in fruitvarieties. For each fruit variety, samples of the fruit were placed ineach of two containers. The fruit samples in one of the containersserved as control samples and samples in the other container served asthe treatment samples. The treatment containers were sealed andconnected to a system which controls and monitors the oxygen levelswithin the container, an example of which is described below and shownin FIGS. 19, 20, 21 and 22. A cross-sectional view of a typicaltreatment container is shown in FIG. 2 and contains a stress monitoringdevice as described above in connection with FIG. 1. The oxygenconcentration was initially lowered to 3% and thereafter the oxygenconcentration was reduced by 0.5% every 12 hours to 0%. After 12 hoursat 0% 0₂, the oxygen concentration was re-established at 3%. Thisprocess created a gradual decrease in the oxygen concentration, ensuringthat the fruit samples were subjected to a dangerous oxygen level forfruit health, followed by a recovery to a healthy oxygen level. For eachfruit variety, the temperature was maintained at approximately 20° C.and CO₂ concentrations within the treatment containers were maintainedat between 0 and 0.5% by placing small bags of hydrated lime in thecontainers which absorbed any CO₂ produced by the respiration of thefruit.

The stress monitoring apparatus was operated at regular intervals andfor each measurement, the intensity of the fluorescence signal wasmeasured and recorded for a number of different values of light sourcelevels. A second order polynomial was fitted to each set of fluorescenceintensity data providing the values of the parameters F_(α), A and B. Anexample of a set of measured data and a second order polynomialregression calculated for the data is shown in FIG. 5. The data is shownas the curve labelled ‘D’ and the polynomial regression is shown by thecurve ‘R’. This data set and regression is also typical of otherexperiments described below.

EXAMPLE 1 Low Oxygen Stress in Apple

FIGS. 6A to 6D show examples of the measured response of apple samplesas the oxygen concentration is progressively reduced. FIGS. 6A, 6B and6C show the variation of parameters F_(α), A and B, respectively, as theoxygen concentration is reduced over time, and FIG. 6D shows thevariation of F_(α) with oxygen concentration. Progressing from higher tolower oxygen concentrations, each of the parameters F_(α), A and Bexhibit little change until hour 72 corresponding to an oxygenconcentration of about 1%. At this concentration, F_(α) and parameter Aincrease abruptly, as shown at ‘start’, whereas parameter B exhibits asharp decrease. F_(α) continues to increase as the oxygen concentrationis lowered further and until the oxygen concentration reaches 0%. As theoxygen concentration is rapidly increased, F_(α) rapidly decreases to asimilar level to that just prior to the onset of the rapid increase.Thus, F_(α) can provide both an indication of the onset of a stresscondition and recovery from a stress condition.

From their respective transition points, both parameters A and Bcontinue to increase and decrease, respectively as the oxygenconcentration is lowered below 1% and both exhibit a rapid change in theopposite direction when the oxygen concentration is quicklyre-established. Thus, both parameters A and B also indicate the onset ofa stress condition and recovery from the stress condition.

EXAMPLE 2 Low Oxygen Stress in Avocado

FIGS. 7A to 7D show examples of the response of avocado samples as theoxygen concentration is reduced. Progressing from higher to lower oxygenconcentrations, the parameter F_(α) initially exhibits little changefollowed by a marked increase at an oxygen concentration of just below1.5%. As the oxygen concentration is lowered further, F_(α) makes asecond abrupt increase at an oxygen concentration of between 0.5 and 1%.At an oxygen concentration corresponding approximately to the secondtransition, parameters A and B also exhibit a marked change, withparameter A decreasing and parameter B increasing. This is the oppositechange to that observed with the apple sample in which parameter aincreased and parameter B decreased. F_(α) continues to increase untilthe oxygen concentration drops to zero and then rapidly decreases to avalue similar to that just prior to the positive transition when theoxygen concentration is quickly reestablished. Parameters A and B alsocontinue to change rapidly as the oxygen concentration is reduced to 0%and then exhibit a sudden change in the opposite direction after theoxygen concentration has remained at 0% for a certain length of time butbefore the oxygen concentration is quickly returned to about 3%.

EXAMPLE 3 Low Oxygen Stress in Banana

FIGS. 8A to 8D show examples of the measured fluorescence response ofbanana samples as the oxygen concentration is progressively reduced.FIGS. 8A, 8B and 8C show the variation of parameters F_(α). A and B,respectively, as the oxygen concentration is reduced over time, and FIG.8D shows the variation of F_(α) with oxygen concentration. The bananasamples used were initially fully green and therefore had an adequatesupply of chlorophyll in the skin to generate fluorescence signals.Progressing from higher to lower oxygen concentrations, F_(α) remainssubstantially constant between about hour 24 and hour 70 as the oxygenconcentration is progressively reduced from about 1.5 to 0.5%. Over thesame time period, parameters A and B fluctuate about a substantiallyconstant value. At an oxygen concentration of approximately 0.5%, F_(α)and parameter A increase abruptly, whereas parameter B exhibits a sharpdecrease, indicating the onset of low oxygen stress in the bananasamples. F_(α) continues to increase as the oxygen concentration islowered further until the oxygen concentration reaches zero percent.Parameter A generally continues to increase and parameter B generallycontinues to decrease as the oxygen concentration is lowered from 0.5%to 0%. As the oxygen concentration is rapidly increased to above thelevel at which the positive transition in F_(α) occurred, F_(α) rapidlydecreases to a similar value to that just prior to the positivetransition, indicating the recovery from stress in the banana samples.As the oxygen concentration is rapidly increased from 0%, the values ofparameters A and B decrease and increase, respectively, to within theirrange of values prior to the onset of low oxygen stress and also providean indicator of the recovery from stress in banana.

FIG. 8A also shows that the value of F_(α) for the control sample ofbananas held at ambient conditions steadily decreases and then tends toa constant value over time. Over this time period, the control bananasamples visibly ripened, thus losing chlorophyll and turning yellow.Therefore, F_(α) provides a sensitive indicator of the loss ofchlorophyll as bananas ripen.

EXAMPLE 4 Low Oxygen Stress in Kiwi Fruit

FIGS. 9A to 9D show examples of the measured fluorescence response ofkiwi fruit with varying oxygen concentration. Referring to FIGS. 9A and9D, F_(α) exhibits little change as the oxygen concentration is reduceduntil about hour 57 corresponding to an oxygen concentration of about0.25%. At this point, particularly as shown in FIG. 9D, F_(α) abruptlyincreases, indicating the onset of low oxygen stress, and continues toincrease as the oxygen concentration is lowered further. As shown inFIG. 9A, F_(α) rapidly decreases at position T₁ as the oxygenconcentration is increased again to about 0.5% at approximately hour 72.As the oxygen concentration is again lowered shortly thereafter, F_(α)again increases rapidly as the oxygen concentration is lowered to 0%,again indicating the onset of low oxygen stress in the kiwi samples. Asthe oxygen concentration is rapidly increased from 0% to above the lowoxygen stress threshold value, F_(α) rapidly decreases to approximatelyits former value before the onset of low oxygen stress. Thus, F_(α)provides a sensitive indicator of both the onset of and recovery fromlow oxygen stress in kiwi fruit. As shown in FIG. 9A, the value of F_(α)for the control sample shows very little change during the period overwhich the tests were conducted and no visible change in the color of thecontrol samples was observed over this period. The values of theparameters A and B fluctuate over the test period but the onset of andrecovery from low oxygen stress is not as readily indicated by theseparameters in the sample under test, as the measurements of Fα.

EXAMPLE 5 Low Oxygen Stress in Mango Fruit

FIGS. 10A to 10D show examples of the measured fluorescence response ofmango samples as the oxygen concentration is varied. Initially, as theoxygen concentration is reduced, F_(α) gradually increases, until atapproximately hour 60 corresponding to an oxygen concentration of about0.75%, the change in F_(α) abruptly increases (particularly as shown inFIG. 10D) as the oxygen concentration is reduced further to 0%,indicating the onset of stressing the health in the mango samples.Thereafter, F_(α) rapidly decreases to approximately its former valuebefore the onset of low oxygen stress as the oxygen concentration israpidly increased again to above the low oxygen stress threshold level,thus indicating the recovery of the mango sample from low oxygen stress.

Parameters A and B fluctuate about a substantially constant value asthe-oxygen concentration is progressively reduced until an oxygenconcentration of approximately 0.75% at which point, parameter Agenerally increases and parameter B generally decreases, and thereforeparameters A and B also provide an indication of the onset of stress. Asthe oxygen concentration is lowered further from 0.5% to 0%, parameter Aexhibits a rapid increase and parameter B exhibits a rapid decrease.This indicates that parameters A and B are particularly sensitive tostress at low oxygen concentrations. After the oxygen concentration hasbeen held at 0% parameter A rapidly decreases and parameter B rapidlyincreases when the oxygen concentration is increased to levels above thelow oxygen stress threshold, indicating the recovery of mango fruit fromlow oxygen stress.

As shown in FIG. 10A, the value of F_(α) for the control samplegradually decreases and tends to a constant value over the test periodand indicates a gradual loss of chlorophyll as the mangoes ripen whichwas also indicated visually as the mango samples changed from green tored or red to yellow over the same period.

EXAMPLE 6 Low Oxygen Stress in Pear

FIGS. 11A to 11D show examples of the measured fluorescence response ofpear samples with varying oxygen concentration. As the oxygenconcentration is reduced, F_(α) initially decreases to about hour 24 atan oxygen concentration of approximately 1.5% and then remainsapproximately constant until about hour 48 corresponding to an oxygenconcentration of about 0.6%. As the oxygen concentration is loweredbelow about 0.6%, F_(α) increases, indicating the onset of low oxygenstress. Above an oxygen concentration of approximately 0.6%, parametersA and B fluctuate about a substantially constant value and parameter Aexhibits a general increase and parameter B a general decrease as theoxygen concentration is reduced below approximately 0.6%. F_(α) andparameter A continue generally to increase as the oxygen concentrationis lowered to 0% and parameter B generally continues to decrease. As theoxygen concentration is rapidly increased from 0% to above the lowoxygen stress threshold, F_(α) and parameter A rapidly decrease toapproximately their respective values prior to the onset of low oxygenstress and parameter B rapidly increases, again returning to its formervalue prior to the onset of low oxygen stress.

FIG. 11A particularly illustrates the sensitivity of F_(α) to smallfluctuations in the oxygen concentration. Between the time period ofhours 24 to 41, the oxygen concentration is switched hourly up and downby about 0.2% between 1.1 and 0.9%, as shown. At these oxygenconcentrations, F_(α) responds by increasing as the oxygen concentrationis reduced and decreasing as the oxygen concentration is increased. Atoxygen concentrations below the low oxygen stress threshold, the oxygenconcentration is occasionally increased during its gradual decrease to0%. Each time the oxygen concentration is increased, F_(α) exhibits acorresponding decrease.

FIG. 11A shows that the value of F_(α) for the control sample graduallydecreases as the pear samples ripen over the test period and indicates aloss of chlorophyll. Over the same period, the pear samples wereobserved to change color from green to yellow.

Monitoring Health in Vegetable Varieties at Low Oxygen Concentrations

The following examples illustrate how embodiments of the presentinvention can be used to detect the onset of low oxygen stress invegetable varieties, and for each test, a similar methodology was usedas described above for the fruit varieties.

For each vegetable variety, samples of the vegetable were placed in eachof two containers. The vegetable samples in one of the containers servedas control samples and samples in the other container served as thetreatments samples. The containers for the control samples were leftopen, whereas the treatment containers were sealed and connected to asystem which controls and monitors the oxygen levels within thecontainer. A cross-sectional view or a typical treatment container isshown in FIG. 2 and contains a stress monitoring device as describedabove in connection with FIG. 1. The oxygen concentration was initiallylowered to 3% and thereafter the oxygen concentration was reduced to 0%at a rate of 0.5% every 12 hours. Thereafter, the oxygen concentrationwas reestablished at 3%. This process created a gradual decrease in theoxygen concentration, ensuring that the vegetable samples were subjectedto a dangerous oxygen level for their health, followed by a recovery toa healthy oxygen level. For each vegetable variety, the temperature wasmaintained at approximately 20° C. and CO₂ concentrations within thetreatment containers were maintained at between 0 and 0.5% by placingsmall bags of hydrated lime in the containers which absorbed any CO₂produced by the respiration of the vegetables.

EXAMPLE 7 Low Oxygen Stress in Cabbage

FIGS. 12A to 12D show examples of the measured fluorescence response ofcabbage samples as the oxygen concentration is progressively reduced.Progressing from higher to lower oxygen concentrations, each ofparameters F_(α), A and B exhibit little change until approximately hour57 corresponding to an oxygen concentration of about 0.2%. At thisoxygen concentration, F_(α) and parameter A increase abruptly, whereasparameter B exhibits a sharp decrease. F_(α) continues to increaserapidly as the oxygen concentration is reduced to 0%. Parameter Acontinues to generally increase and parameter B generally continues todecrease as the oxygen concentration is reduced to 0%. When the oxygenconcentration is rapidly re-established to above 1.5%, F_(α) rapidlydecreases to a value slightly above its former value just prior to theonset of low oxygen stress. At a time prior to the re-establishment ofthe oxygen concentration, parameter A decreases and parameter Bincreases to approximately their former values just prior to the onsetof low oxygen stress.

EXAMPLE 8 Low Oxygen Stress in Green Pepper

FIGS. 13A to 13D show examples of the measured fluorescence response ofgreen pepper samples as the oxygen concentration is varied. Progressingfrom higher to lower oxygen concentrations, parameters F_(α) and Ainitially exhibit a gradual increase, whereas parameter B shows agradual decrease.

At approximately hour 40, corresponding to an oxygen concentration ofabout 0.8%, the positive change in both F_(α) and parameter A increasesand parameter B abruptly decreases. F_(α) continues to increase as theoxygen concentration is lowered to just above 0% at approximately hour72. Within the same interval, parameter A exhibits a marked increase andparameter B shows a marked decrease. After hour 72, with continuedreduction of the oxygen concentration to 0%, F_(α) and parameter A bothdecrease to approximately to their former values prior to the onset oflow oxygen stress and parameter B rapidly increases, again to its formervalue prior to the onset of low oxygen stress.

Significant deterioration of the green pepper samples had occurredduring the test period and this may account for the decrease in Fα andparameter A and the increase in parameter B prior to there-establishment of a healthy oxygen concentration.

EXAMPLE 9 Low Oxygen Stress in Iceberg Lettuce

FIGS. 14A to 14D show examples of the measured fluorescence response oficeberg lettuce samples as the oxygen concentration is progressivelyreduced. Initially, F_(α) gradually increases as the oxygenconcentration is reduced until approximately hour 62 corresponding to anoxygen concentration of just above 0% at which point the positive changein F_(α) exhibits a marked increase. Initially, parameters A and Bremain relatively constant as the oxygen concentration is reduced againuntil about hour 62, where parameter A exhibits an abrupt increase andparameter B exhibits a marked decrease, indicating the onset of lowoxygen stress. F_(α) generally continues to increase until the oxygenconcentration reaches 0%. When the oxygen concentration is rapidlyreestablished to a value above the onset of low oxygen stress, F_(α)rapidly decreases to approximately its value prior to the onset of lowoxygen stress, indicating recovery of the iceberg lettuce sample fromlow oxygen stress.

EXAMPLE 10 Low Oxygen Stress in Romaine Lettuce

FIGS. 15A to 15D show examples of the measured fluorescence response ofromaine lettuce as the oxygen concentration is progressively reduced.Initially, F_(α) and parameter A exhibit a small gradual increase as theoxygen concentration is lowered until approximately hour 60corresponding to an oxygen concentration of just above 0%. Over the sameperiod, parameter B exhibits a small gradual decrease. At approximatelyhour 60, the positive change in both F_(α) and parameter A exhibit amarked increase indicating the onset of low oxygen stress, and parameterB exhibits an abrupt decrease, again indicating the onset of low oxygenstress. As the oxygen concentration is reduced further to 0%, F_(α)continues its relatively rapid increase, parameter A continues generallyto increase and parameter B continues generally to decrease. When theoxygen concentration is again re-established to a healthy level atapproximately hour 84, F_(α) rapidly decreases to approximately itsformer value prior to the onset of low oxygen stress indicating recoveryof the romaine lettuce sample from low oxygen stress and parameters Aand B exhibit a marked decrease and increase, respectively towards theirformer values prior to the onset of low oxygen stress, again indicatingrecovery of the romaine lettuce samples from low oxygen stress. As shownin FIG. 15A, the value of F_(α) of the control sample remains relativelyconstant over the test period.

Monitoring Health in Chlorophyll Containing Matter in Response to HighCarbon Dioxide Concentrations

The following example illustrates how embodiments of the presentinvention can be used to detect the onset of high CO₂ stress inchlorophyll containing matter. In the example, the fluorescence responseof cabbage samples was measured with varying CO₂ levels for twodifferent oxygen concentrations.

The apparatus for monitoring high CO₂ stress in this example is the sameas that used to monitor low oxygen stress in various fruit and vegetablevarieties, described above. Cabbage samples to be treated with high CO₂levels were placed in sealed containers connected to an atmospherecontrol system for controlling the various levels of nitrogen, oxygenand carbon dioxide. Control samples were also placed in containers andmaintained at ambient conditions. CO₂ concentrations in the treatmentcontainers initially started at 0% and were increased by 2% every 12hours until the concentration had risen to 12%. The CO₂ concentrationwas then lowered back to 0%. The temperature was maintained atapproximately 20° C. over the test period.

EXAMPLE 11 High CO₂ Stress in Cabbage

FIGS. 16A to 16E show examples of the measured fluorescence response ofcabbage samples with varying CO₂ concentrations. The test shown in FIG.16A was performed at an oxygen concentration of 4% and the test shown inFIGS. 16B to 16E was performed at an oxygen concentration of 1.5%.

FIG. 16A shows the response of F_(α) as the CO₂ level is varied,together with F_(α) for the control sample. Initially, as the carbondioxide level is increased, F_(α) of the treatment sample remainsrelatively constant until approximately hour 24 when the CO₂concentration is increased from about 5.5% to about 9%. At the sametime, F_(α) exhibits a noticeable increase, indicating the onset of highCO₂ stress in the cabbage sample. Shortly after hour 24, the CO₂concentration increases more slowly to about hour 36 and F_(α) steadilyincreases over the same period. At hour 36, the carbon dioxideconcentration is again rapidly increased from just below 10% to 12% andis maintained at this level until about hour 72. Shortly after thisabrupt increase in carbon dioxide level, F_(α) increases at a fasterrate, indicating that the cabbage samples are experiencing increasingstress as the carbon dioxide levels are increased further. F_(α)continues to increase at this new accelerated rate over the period atwhich the CO₂ level is held at 12%.

At hour 72, the CO₂ concentration is rapidly reduced to 0% and at thesame time F_(α) decreases, indicating recovery of the cabbage samplesfrom CO₂ stress. It is observed that the value of F_(α) does not returnto its former value just prior to the onset of high CO₂ stress duringthe 24 hour period following the rapid return of the CO₂ concentrationto 0%, which may indicate that permanent physiological change hasoccurred within the cabbage samples. The value of F_(α) for the controlsample varies very little over the test period. Parameters A and B whichwere measured for the treatment sample over the test period wererelatively stable and showed little change in response to the varyingCO₂ concentration.

FIG. 16B shows the variation of F_(α) with CO₂ concentration at anoxygen concentration of about 1.5%, together with the variation of F_(α)for the control sample. As the carbon dioxide level is increased from0%, F_(α) initially changes very little, first making a small decreaseand then a very gradual increase until about hour 24 corresponding to acarbon dioxide level of about 3%. As the carbon dioxide level isincreased above about 3%, the positive change in F_(α) increases,indicating the onset of high carbon dioxide stress. F_(α) continues toincrease at approximately the same rate with increasing carbon dioxideconcentration until about hour 60 and thereafter begins to level off.For this sample, the onset of high carbon dioxide stress occurred at alower carbon dioxide concentration than for the sample shown in FIG. 16Awhich may be attributed to differences in the samples tested or to anincrease sensitivity to high carbon dioxide concentrations at loweroxygen levels.

The carbon dioxide level is reduced relatively quickly from about hour84 and it is observed that F_(α) begins to decrease before the carbondioxide level is reduced. In comparison to the sample test results shownin FIG. 16A, the cabbage samples of FIG. 16B are held in relatively highcarbon dioxide concentrations over a longer period of time and therelatively small decrease in F_(α) towards the end of the test mayindicate that the cabbage sample has sustained permanent physiologicalchange.

Referring to FIGS. 16C and 16D, parameter A steadily decreases as thecarbon dioxide concentration is increased whereas parameter B steadilyincreases and at a carbon dioxide concentration of about 7%, parameter Apasses through a minimum and thereafter gradually increases as thecarbon dioxide concentration continues to increase and at the samecarbon dioxide concentration (about 7%) parameter B passes through amaximum and thereafter gradually decreases with increasing carbondioxide concentration. Thus, in addition to F_(α), parameters A and Bare both sensitive to CO₂ levels at lower oxygen concentrations and canbe used to detect the presence of CO₂ and/or to provide an indication ofthe level of CO₂ and may be used to provide a warning of when CO₂ levelsexceed concentrations for a healthy environment.

Detecting the Reaction of Chlorophyll Containing Matter to TemperatureChanges

The following example illustrates how embodiments of the presentinvention can be used to detect how chlorophyll containing matterresponds to temperature changes. In this example, chlorophyllfluorescence monitoring devices as described above and shown in FIG. 1were used to monitor the fluorescence response of unripened bananasamples as the temperature of the room in which the samples were storedwas varied. The banana samples which each consisted of a cluster ofthree bananas were placed in fruit kennels and their fluorescenceresponse was monitored hourly as the room temperature was lowered from15° to 3° in 3° increments every 24 hours. The fluorescence response ofa control sample of bananas was also monitored using a fluorescencemonitoring device as shown in FIG. 1 in a room at 22° C.

EXAMPLE 12 Monitoring Temperature Response in Banana Samples

FIG. 17A shows a graph of Fα as a function of time of a banana sampleheld in ambient atmosphere at 22° C. Over the first 24 hour period,F_(α) is seen to decrease relatively rapidly and then decrease moreslowly over the following 2½ days. This decline in F_(α) over the testperiod results from the banana ripening and losing chlorophyll as thebanana sample turns from green to yellow at the end of the test periodwhen the banana sample is fully ripened. Thus, embodiments of thepresent invention may be used to detect the loss of chlorophyll inchlorophyll containing produce, for example as the produce ripens. Infurther embodiments of the present invention, the detection of loss ofchlorophyll resulting, for example in ripening of a stored product maybe used to control one or more environmental parameters to reduce theloss of chlorophyll or the rate of loss of chlorophyll and slow theripening of the produce.

FIG. 17B shows the variation of F_(α) with temperature as thetemperature is lowered incrementally from 15° C. to 6° C. over a fourday period and is then returned to 15° C. for a further 24 hour period.F_(α) remains relatively constant during the first and second 24 hourperiods at temperatures of 15 and 12° C., respectively. At about hour 60during the third 24 hour period at a temperature of 9° C., F_(α) beginsto decrease as indicated at D₁. F_(α) continues to decrease over thenext 24 hour period and at about hour 90 during the fourth 24 hourperiod at a lower temperature at 6° C., F_(α) decreases at a higherrate, as indicated at D₂. F_(α) continues to decrease at this higherrate until the temperature is quickly increased to above 15° C. at hour96. At this point, the decline in F_(α) ceases and F_(α) remainsrelatively constant over the next 12 hour period at a temperature of 15°C. before decreasing again at the same temperature The particular bananasamples used in this test remained green over the five day test periodand beyond but ripened eventually.

The results indicate that Fα can be used to detect the response ofchlorophyll containing produce to both decreasing and increasingtemperatures. Although the previous results show that the onset ofstress in chlorophyll containing produce due to changes in environmentalparameters such as oxygen and carbon dioxide concentrations is signifiedby an increase in Fα, the processes which define the fluorescenceresponse of chlorophyll due to temperature changes are likely to bedifferent and therefore invoke a different response in Fα. Thus, Fα canbe used to monitor temperature-induced reactions in chlorophyllcontaining produce and may be used to monitor independently temperaturechanges in the environment in which the produce is stored and mayfurther be used to control the temperature of the environment.

FIG. 17C shows the variation of Fα with temperature for a differentbanana sample. Over the first 12 hour period at a temperature of 15° C.,Fα decreases slightly as the temperature is lowered to 12° C. beforedecreasing again to a value which remains relatively constant over thenext three 24 hour periods until hour 96 when the temperature is quicklyraised from 3° C. to about 20°0 C. As the temperature is rapidlyincreased, Fα rapidly decreases and then increases again to a valuebelow its previous value and thereafter decreases steadily as thetemperature is held at about 15° C. Thus, Fα can detect the response ofchlorophyll containing produce to thermal shock. At about hour 110, thetemperature rapidly fluctuates, initially decreasing to about 7° C. thenincreasing to about 17° C. and finally settling again at about 15° C.Again, Fα responds to this rapid temperature fluctuation by firstdecreasing and then rapidly increasing within the same time period asthe temperature fluctuation before resuming a gradual decline when thetemperature returns to 15° C. Thus, Fα may be used to detect suddentemperature changes of the environment in which chlorophyll containingmatter is stored. Detection of such an event may be used to warn anoperator or control system so that any appropriate action can be taken.

Monitoring Moisture Stress in Chlorophyll Containing Matter

The following example illustrates how embodiments of the presentinvention can be used to detect the onset of stress due to moisture lossin chlorophyll containing matter. In this example, the fluorescenceresponse of the leaves of strawberry plants was monitored as themoisture content of the leaves was lost.

In this example, mature potted strawberry plants which had beenmaintained in a greenhouse and watered regularly to ensure good planthealth were used. Individual plants were transferred from the greenhouseto a laboratory to be monitored by fluorescence monitoring devices.Three fluorescence monitoring devices as described above and shown inFIG. 1 were placed over three individual intact strawberry leaves tomonitor each leaf separately. The leaves were taped to a plastic sheetto prevent them from moving and the fluorescence monitoring devices weremounted over each leaf at a distance of 7 cm from the leaf surface.

The fluorescence monitoring devices, sampled the leaf fluorescence every15 minutes throughout a three day period. After approximately 24 hoursof measurements, in order to obtain a baseline reading, two of the threeleaves were cut from the strawberry plant. The third leaf was keptintact and at this point the plant was also watered. The stem of one ofthe cut leaves was placed in a beaker of water to provide additionalmoisture to keep the leaf properly hydrated. The other cut leaf and itsstem were simply exposed to air and received no further water for theremaining two days of the test period. This cut leaf with water additionwas used to check if the act of cutting the stems from the plant causeda significant fluorescence change in the leaf samples. After cutting,the fluorescence monitoring devices monitored the samples for anadditional two days and after three days the plant was removed andreplaced with a new healthy plant from the greenhouse and the processrepeated.

EXAMPLE 13 Moisture Stress in Strawberry Plants

FIGS. 18A and 18B show examples of the fluorescence response of theleaves of two strawberry plants. During the first 24 hour period whennone of the monitored leaves were cut, the fluorescence response of allthree leaves is relatively stable. F_(α) for the cut unwatered leaf ofthe plant of FIG. 18A begins to increase shortly after the leaf is cutindicating the onset of stress due to lack of moisture, and continues toincrease at a steady rate over about the next 24 hour period. During thethird 24 hour period, i.e. at about 3000 minutes, F_(α) for the cutunwatered leaf abruptly increases, indicating a more severe increase inmoisture loss induced stress. Over the second and third 24 hour periodsF_(α) for the cut, watered leaf and the uncut leaf remain relativelyconstant.

F_(α) for the cut, unwatered leaf of the plant of FIG. 18B initiallyremains relatively constant after the leaf is cut, then decreasesslightly during the second 24 hour period and subsequently at about thestart of the third 24 hour period increases abruptly indicating theonset of low moisture stress. Over the second and third 24 hour periods,F_(α) for the cut, watered leaf and the uncut leaf exhibit littlechange. The visual analysis of the leaves after each test showed thatthe intact and cut, watered leaves were still healthy but the cut,unwatered leaves had become quite dry. Embodiments of this method ofmeasuring stress due to moisture loss in chlorophyll containing matterusing chlorophyll fluorescence may be applied to any suitable plantsincluding both rooted and cut plants and may be used in indoor oroutdoor applications for detecting moisture stresses in plant materials.For example, the technique may be used for plants in residential andcommercial buildings, greenhouses or in the field.

Embodiments of the stress monitoring method and apparatus may be used todetect the onset of stress or physiological change in any chlorophyllcontaining matter which exhibits a detectable transition in the changeof fluorescence intensity level, or in a parameter derived from theintensity level which is sensitive to stress or physiological change.

The monitoring apparatus and method may be used to monitor the healthand well-being of living plants, cultivars, fruits and vegetables sothat appropriate action can be taken to maintain a healthy condition.The apparatus and method may be used to provide a warning that anenvironmental parameter is at an incorrect value and needs to bechanged. For example, the apparatus and method could be applied tocontrolled atmosphere storage to alert an operator that the oxygenconcentration is too low or the concentration of carbon dioxide is toohigh. The apparatus and method can also be applied to determine theoptimum environmental conditions for storing fruits and vegetablesand/or to dynamically control the environment. Examples of how theapparatus and method may be applied to determine the optimum oxygenconcentration for storing fruits and vegetables and/or for controllingthe relative gas concentrations in the storage environment will now bedescribed with reference to FIGS. 19 to 31.

Referring to FIG. 19, a combined controlled atmosphere and chlorophyllfluorescence measurement system according to an embodiment of thepresent invention, generally indicated at 1, comprises a plurality ofstorage jars 3, for containing one or more fruit or vegetable sample, agas control system 5 for controlling the relative concentrations ofdifferent gases contained within each storage jar 3 and a fluorescencemeasurement system 7 for measuring the level of chlorophyll fluorescenceemitted from the fruit or vegetable samples. Gas canisters 9 and 11,serving as sources of carbon dioxide and nitrogen gas, respectively, areconnected to the gas control system 5. A computer 13, for example a PC,controls the operation of the gas controller 7 to regulate changes inthe concentrations of gases in each storage jar 3, and collects andanalyzes data from the fluorescence measurement system 7. The computer13 may include user interfaces such as a visual display and keyboard 14.

Referring to FIG. 20, the gas control systems includes a gas controller15 which receives carbon dioxide (C0 ₂) and nitrogen (N₂) from canisters9,11 and air from the atmosphere, and feeds a specified amount of aselected gas to a particular sample jar. The sample jar is selectedunder the control of respective gas inlet valves, collectively shown asa valve bank 17, connected to the gas inlet port in each sample jar 3.Each sample jar 3 has a gas outlet port connected to a gas outlet valve,also shown as being grouped within the valve bank 17, which controls theflow of gas from each sample jar to gas analyzers, collectively shown at19, for analyzing the content of various gases within the sample jars 3.In this embodiment, the gas analyzers measure the levels of carbondioxide, oxygen and optionally ethanol.

Referring to FIG. 21, which shows an example of an arrangement of inletand outlet gas control valves in more detail, each of the inlet valves21 is connected to a feed line 23 which supplies gas from the gascontroller to a particular jar selected according to which gas inletvalve is open. Each gas outlet valve 25 is connected to a common gasfeed line 27 which supplies gas from a jar, selected according to whichoutlet valve is opened, to the gas analyzers 19. The inlet and outletvalves 21, 25 are preferably capable of being actuated electrically sothat they can be opened and closed automatically under the control ofthe computer 13 (FIG. 19). Generally, when the gas in a particular jaris being sampled or its gas content changed, both the inlet and outletvalves of that jar particular are opened and the inlet and outlet valvesof all other jars are closed.

Referring to FIG. 22, the gas analyzer system 19 includes a filter 29connected to receive a gas sample from a selected sample jar and forremoving any airborne particles, a pressure sensor 31 for measuring thegas pressure in the selected sample jar, an oxygen sensor 33, a carbondioxide sensor 35 and, optionally, an ethanol sensor 37, for measuringthe oxygen, carbon dioxide and ethanol content, respectively, of the gasin a specified jar. During gas sampling, a portion of the gas is drawnfrom the jar by a pump (not shown), is passed through each of the gassensors and returned to the sample jar after analysis.

Between each jar sampling, the analyzers are purged by flushing withnitrogen gas to avoid cross contamination between different sample jars,and the purged gas is subsequently vented after leaving the gasanalyzers.

To change the relative concentrations of the gases within a sample jar,a controlled amount of air, nitrogen or carbon dioxide is drawn into thejar by a pump. For example, to increase the oxygen content, air ispumped into the jar, whereas to decrease the oxygen level, nitrogenand/or CO₂ is pumped into the jar. In either case, the gas inlet andoutlet valves of the selected jar are opened and gas is drawn from thejar, passed through the gas analyzers and returned to the jar. The gascontroller 15 introduces the selected additional gas into the gas streamwhich subsequently mixes with the gases contained in the jar. A pumpcontinues to draw gas from the sample jar and analyzes the gas samplefor oxygen, CO₂ and ethanol, if required. When the desired gasconcentration is obtained, the selected gas supply is stopped and thegas inlet and outlet valves closed. During any gas addition to thesystem, excess gas is vented so that the gas pressure remainssubstantially constant.

FIGS. 23 and 24 show an embodiment of a fluorometer used to stimulateand measure chlorophyll fluorescence from fruit or vegetable samples ineach jar. The fluorometer 31 comprises three light source/sensingstations 33,35,37 spaced equally around and at equal distances from asample jar 3, in a triangular arrangement, as shown in FIG. 23. Thethree light source/sensor station arrangement enables morerepresentative measurements of the sample to be made. Referring to FIG.24, each station comprises a rectangular array of four light emittingdiodes (LEDs) 39,41,43,45, a source of white light 47 positioned withinthe rectangular array of LEDs and a photodiode 49, all mounted on asupport panel 51. The light emitting diodes in each station serve tostimulate a minimal or dark fluorescence Fo in the chlorophyll of thefruit or vegetable sample. In one embodiment, the LEDs generate lowintensity red light at wavelengths of about 660 nanometers with anintensity at the sample surface of generally less than 10 μmol.m⁻².s⁻¹.Light from the light emitting diodes is such as to cause chlorophyllfluorescence and is capable of stimulating fluorescence in the regimewhere all photosystem II reaction centres are open while thephotosynthetic membrane is in the non-energized state, i.e. to measure aminimal fluorescence signal Fo. The photodiode 49 in each stationdetects the intensity of the fluorescence signal Fo. emitted by thechlorophyll which is recorded by the computer 13. The white light source47 in each station serves to stimulate a maximal fluorescence signal Fmdefined as the fluorescence intensity emitted when all photosystem IIreaction centres are closed and all non-photochemical quenchingprocesses at a minimum. In one embodiment, the white light source is a250 Watt Tungsten filament bulb. Again, the photo diode 49 in eachstation detects the maximal fluorescence signal Fm which is againrecorded by the computer 13.

Methods of determining an optimum oxygen level in which to store fruitsand vegetables, according to embodiments of the present invention willnow be described with reference to FIGS. 25A to 30B.

A fruit or vegetable sample is placed in a sample jar which is thensealed so that gas may only be introduced or drawn from the jar via theinlet and outlet ports under the control of the system control valves.Fruit or vegetable samples may additionally be placed in some or all ofthe other jars which are also subsequently sealed. The starting pointoxygen concentration is then established in each jar and may range forexample from 3 to 21 percent as required. A starting point of low oxygenconcentrations may be established for fruit and vegetable samples whichare known to be capable of tolerating low oxygen level atmosphereswithout being damaged by low oxygen stress. A measurement of the minimalfluorescence intensity Fo may be made at the oxygen concentrationstarting point, and, as described above, this is achieved by activatingthe light emitting diodes in each station of each fluorometer toirradiate portions of the surface of the fruit or vegetable sample tostimulate minimal chlorophyll fluorescence and detecting thefluorescence signal emitted from the chlorophyll by means of thephotodiodes. A maximal fluorescence measurement may also be made byactivating the white light source and, again, detecting the maximalfluorescence intensity by means of the photodiodes. The initial oxygenconcentration and values of minimal and maximal fluorescence intensitiesare then recorded by the computer.

The oxygen concentration in one or more sample jars is thenprogressively reduced at a rate, for example 0.2%/h, by introducingadditional quantities of nitrogen into the jar using, for example, thegas control system described above. A measurement of the minimalfluorescence intensity signal Fo and optionally the maximal fluorescenceintensity signal Fm is made and recorded at each oxygen level.

For each measured oxygen concentration, a rolling average value of theminimal fluorescence intensity Fo is calculated based on the fiveprevious and the current values of Fo. The difference between thecurrent Fo value and the current rolling average value of Fo at eachmeasured oxygen concentration is also calculated to give the change ofthe current Fo value from the current rolling average, and thefractional or percentage change between the current Fo value and thecurrent rolling average value is then calculated. A current averagefractional or percentage change is then calculated from the fiveprevious values and the current value of the fractional or percentagechange. The ultra low oxygen (ULO) threshold is determined as the oxygenlevel below which six consecutive values of the average fractional orpercentage change in Fo is greater than 0.01 or 1%, respectively.

If Fm is measured, the value Fv/Fm, where Fv=Fm−Fo, may also becalculated.

Examples of applications of the above described method to determine theoptimum oxygen concentration for controlled atmosphere storage ofvarious fruit samples are described below.

EXAMPLE 1

The method according to the above described embodiment was applied to anapple sample. FIG. 25A shows a graph of the variation of Fo, Fv/Fm andpercentage oxygen concentration with time, and FIG. 25B shows a table ofmeasured values of percentage oxygen content, minimal and, maximalfluorescence intensities Fo and Fm, calculated values of the rollingaverage of Fo, the change or difference between the current measured Foand rolling average Fo values, the calculated percentage change and theaverage percentage change.

Referring to FIG. 25A, the oxygen concentration was progressively andgradually reduced and the minimal and maximal chlorophyll fluorescenceintensities were measured every hour along with the oxygenconcentration. The graph and table show that Fo remains substantiallyconstant until hour 65, after which time Fo steadily increases and theaverage percentage change in Fo for the next consecutive 6 hours, i.e.from hour 66 to hour 71 is greater than 1%. The increase in Fo and inits average percentage change indicates a precipitation of low oxygenstress and the onset of possible damage. The optimum oxygenconcentration for storing any fruit or vegetable is the lowest valueabove that which would otherwise cause damage to the product by lowoxygen stress. In the present embodiment, the optimum oxygenconcentration is determined as that just before the average percentagechange reaches a value of greater than 1% for six consecutive readings,which in the present case 2.44%.

Referring again to FIGS. 25A and 25B, Fo continues to increase as theoxygen concentration continues to decrease below the optimum thresholdand the change in Fo becomes more severe from hour 108 onwards, as shownby the increase in the average percentage change from this point in thetable of FIG. 25B.

FIGS. 25A and 25B also show that Fv/Fm progressively decreases as theoxygen concentration is reduced and then begins to decrease at a higherrate at a point in time and oxygen concentration which closelycorresponds to the optimum oxygen concentration at which the change inFo begins to increase. As the oxygen concentration is lowered stillfurther, Fv/Fm decreases more abruptly at a position in time and at avalue of oxygen concentration closely corresponding to that at which Foexhibits a more severe increase.

FIG. 25A also shows that when the oxygen concentration is suddenlyincreased from its lowest level to a value above the optimum thresholdlevel, Fo decreases at a similar rate to a value closely correspondingits former values above the optimum oxygen concentration threshold.Fv/Fm also increases at a similar rate, returning to levels similar tothose prior to the onset of the increased change at the optimum oxygenconcentration level. This latter behaviour indicates that no permanentdamage was sustained by the fruit in the method of determining theoptimum oxygen concentration threshold and that the methodadvantageously provides a means for determining this value withoutdestroying the sample.

EXAMPLE 2

FIG. 26A is a graph showing the variation of Fo and Fv/Fm for a kiwifruit sample as the oxygen concentration in which the sample is placedis progressively reduced. FIG. 26B shows part of the data plotted inFIG. 26A in tabulated form, and in addition the rolling average of Fo,the change between the current value of Fo and its corresponding rollingaverage, the percentage change and the average percentage change in Fo,the maximal fluorescence Fm and the calculated value of Fv/Fm. As theoxygen concentration is progressively lowered, Fo remains substantiallyconstant until a time corresponding to hour 36 at which Fo increases andthe average percentage change in Fo exceeds 1% and remains above 1% forthe next consecutive six points, and beyond, as the oxygen concentrationcontinues to be reduced. This increase in the change of Fo indicates theonset of low oxygen stress in the kiwi fruit sample. The optimal oxygenconcentration threshold may be determined as the oxygen concentrationjust prior to the onset of this increase in the change of Fo, which inthe present case is 2.4%.

Returning to FIG. 26A, it can be seen that, initially, at the higheroxygen concentrations, Fv/Fm steadily decreases between hours 0 and 35(ignoring the periodic fluctuations) and at a time and the same value ofoxygen concentration as the change in Fo has started to increase, thechange in Fv/Fm also starts to increase. Thus, measuring the change inFv/Fm may also be used to determine the optimal oxygen concentrationthreshold.

As the oxygen concentration is progressively reduced below the oxygenthreshold measured at hour 35, Fo continues to increase whereas Fv/Fmcontinues to decrease, both indicating a continued increase in lowoxygen stress in the sample. As the oxygen level is suddenly increasedjust before hour 120, Fo is seen to decrease and Fv/Fm is seen toincrease at a similar rate towards their former, pre-oxygen stresslevels.

EXAMPLE 3

FIGS. 27A and 27B show the variation in Fo and Fv/Fm for a mango sampleas the oxygen concentration of the atmosphere in which the sample isplaced is progressively reduced. The results indicate that initially theminimal fluorescence intensity Fo remains relatively constant withdecreasing oxygen concentration and then at a time corresponding to hour200, Fo begins to increase such that its average percentage changeexceeds 1% for at least the next consecutive six points. The optimumoxygen concentration threshold for the mango sample may then bedetermined from this transition of the change in Fo as 0.4%: the oxygenconcentration at hour 199 just prior to the point at which the averagepercentage change in Fo continuously exceeds 1%.

As can been seen from FIG. 27A, Fv/Fm steadily decreases with decreasingoxygen concentration and then at a point corresponding to that at whichthe change in Fo increases, Fv/Fm exhibits a precipitous drop alsoindicating the onset of low oxygen stress in the mango sample.

EXAMPLE 4

FIGS. 28A and 28B show the variation in minimal fluorescence intensityFo and FV/Fm for a pear sample as the oxygen concentration of theatmosphere in which the pear sample is placed is progressively reduced.The measurements indicate that, initially, Fo remains relativelyconstant with decreasing oxygen concentration until a time correspondingto hour 28 at which Fo starts to progressively increase, indicating theonset of low oxygen level stress occurring in the sample. At this point,the average percentage change in Fo exceeds and continues to exceed 1%,as shown in the table of FIG. 28B. The optimal oxygen concentrationthreshold is determined on the basis of this change in Fo and forexample may be established as the oxygen concentration of 2.87% justbefore the onset of the increase in Fo which also corresponds to theoxygen concentration just before the average percentage change in Focontinuously exceeds 1% for at least the next six consecutive points.

Returning to FIG. 28A, Fv/Fm initially exhibits a steady, substantiallymonotonic decrease (ignoring periodic fluctuations in the data) as theoxygen concentration is progressively reduced and then at a point whichsubstantially corresponds the point at which Fo increases, the decreasein Fv/Fm markedly accelerates.

EXAMPLE 5

FIGS. 29A and 29B show the variation in the minimal fluorescenceintensity Fo and Fv/Vm for an avocado sample as the oxygen concentrationof the atmosphere in which the sample is placed, is progressivelyreduced. Initially, Fo remains substantially constant with decreasingoxygen concentration until, at a time corresponding to hour 86, Fobegins to increase, indicating the onset of low oxygen stress in thesample. The optimal oxygen threshold may be determined on the basis ofthis increase in Fo and established as for example 1.3% corresponding tohour 85, just prior to the average percentage change continuouslyexceeding 1% for six consecutive readings.

Referring to FIG. 29A, Fv/Fm initially decreases at a steady rate as theoxygen concentration is progressively lowered (allowing for thefrequent, intermediate fluctuations in data) and at a point closelycorresponding to that at which Fo increases, Fv/Fm exhibits aprecipitous drop as the oxygen concentration is lowered further.

EXAMPLE 6

FIGS. 30A and 30B show the variation in minimal fluorescence intensityFo and Fv/Fm for a banana sample as the oxygen concentration of theatmosphere in which the sample is held, is progressively reduced.Initially, at higher oxygen concentrations Fo steadily decreases until atime corresponding to hour 11 at which Fo starts to increase, indicatingthe onset of low oxygen stress in the sample. The optimal oxygenconcentration threshold for storing the banana sample may be determinedon the basis of the transition in the change in Fo and for example maybe established as an oxygen concentration of 0.61% corresponding to hour10. Below this oxygen concentration, the average percentage change in Foexceeds 1% for at least six consecutive readings as the oxygenconcentration is lowered further.

Referring to FIG. 30A, at higher oxygen concentrations, Fv/Fm initiallyexhibits a steady downward progression (allowing for periodicfluctuations in the data) and then, at a point substantiallycorresponding to that at which Fo begins to increase, the decrease Fv/Fmsuddenly accelerates.

The above examples 1 to 6 illustrate that the method according toembodiments of the invention may be used to detect the onset of lowoxygen stress in chlorophyll containing produce and to determine thespecific optimal oxygen concentration threshold which minimizesrespiration without causing damage, for a given product. Advantageously,this allows the time over which the product can be stored withoutdeterioration of quality to be maximized or, if the product is to bestored for a period of time less than the maximum, the method allows anydeterioration in the product to be minimized over that time andtherefore the quality of the product after the storage time to beimproved in comparison to products stored under existing storagetechniques.

In any of the above examples, and in practising embodiments of themethod for mass fruit and vegetable storage, the optimal oxygenthreshold may be determined more accurately by making additional,intermediate measurements of Fo and/or Fv/Fm around the oxygenconcentrations where the changes in these parameters begin to increase.

In another embodiment of the present invention, a method of controllingthe oxygen concentration in an atmosphere in which chlorophyllcontaining fruit or vegetables are stored comprises varying the oxygenconcentration and monitoring the minimal fluorescence intensity Foemitted by the stored produce and determining from the measuredintensity, the oxygen concentration at which the onset of low oxygenstress in the produce occurs, preferably controlling the oxygenconcentration to minimize respiration of the produce without causing lowoxygen damage and immediately or after some time has elapsed, againreducing the oxygen concentration to determine any change in the optimumoxygen concentration threshold, and adjusting the oxygen concentrationbased on any change in the optimum threshold value.

The inventors have found that during storage, a product's tolerance tolow oxygen levels before the onset of low oxygen stress can increasewith time, so that the optimum oxygen concentration threshold for aparticular product can decrease during the storage period. Therefore,the present method allows the storage time to be extended further byperiodically reducing the oxygen concentration and monitoring theminimal fluorescence intensity Fo to determine any change in the optimumoxygen concentration threshold. This method effectively uses the storedproduct to indicate the lowest oxygen concentrations it can tolerate atvarious times during the storage period so that the oxygen concentrationcan be dynamically adjusted to provide the optimum conditions formaximizing the storage period for the particular product. An example ofthis method applied to the storage of apples will now be described underexample 7.

EXAMPLE 7

The following test was applied to McIntosh cultivars of Marshall and RedMax apples which were held under controlled atmosphere (CA) conditionsfor four months. A first sample of the apples were placed storageconditions in which the oxygen and carbon dioxide concentrations wereheld constant over the 4 month storage period at 2.5% 0₂ and 4.5% C0₂. Asecond sample of the apples were placed under storage conditions inwhich the oxygen concentration was periodically stepped down based onwhat the fluorescence intensity emitted by the fruit indicated was thelowest oxygen concentration they could withstand without inducingdamage.

After a period of four months, both samples were removed from storageand subjected to firmness and taste tests, as follows. Immediately afterthe four month storage period, samples were placed in cold storage forfourteen days at 3° C. and thereafter tested for firmness. The resultsfor two Marshall and one Red Max apple stored in each of the constant(standard) and stepped oxygen concentration conditions are shown inTable 7 of FIG. 31. The results indicate that the apples stored in thestepped controlled atmosphere were on average 1.49 pounds firmer thanthose stored in the constant controlled atmosphere.

The samples of the apples stored in each of the constant and stepped CAstorage conditions were taste tested by twelve panellists. The resultsshow that for Marshall MacIntosh apples, 40% of the panellists expresseda preference for the apples stored under the stepped controlledatmosphere conditions, whereas 25% expressed a preference for the applesstored under the standard conditions.

For the Red Max cultivar, 90% of the panellists expressed a preferencefor those apples stored under stepped conditions, whereas no panellistsexpressed a preference for those stored under standard conditions.

Both the Marshall McIntosh and Red Max were also tested for the presenceof off-flavours. The results show that for Marshall McIntosh, 70% ofpanellists detected no off-flavours in the samples stored under thestepped conditions whereas 50% of panellists detected no off-flavours inthe apples stored under standard conditions. For Red Max, 90% ofpanellists detected no off-flavours in the samples stored under steppedconditions, whereas 50% of panellists detected no off-flavours in theRed Max McIntosh samples stored under the standard conditions.

These results collectively indicate that measurements of chlorophyllfluorescence on stored fruit allows the optimum oxygen concentrationthreshold to be found and that dynamically adjusting the oxygenconcentration to track the optimum threshold as the threshold variesover the storage period better preserves the fruit quality.

An embodiment of an apparatus for tracking the optimum concentrationthreshold during the storage of fruit or vegetables comprises means fordetecting an increase in the change in fluorescence intensity withdecreasing oxygen concentration, means for controlling the oxygenconcentration to a level corresponding to the increase in fluorescenceintensity and means for periodically reducing the oxygen concentrationand re-establishing the optimal oxygen concentration based on anyincrease in the change of fluorescence intensity as the oxygenconcentration is lowered. The oxygen concentration and fluorescencemeasurements may be controlled by a microprocessor under the control ofa suitable program.

In another embodiment of the method of determining an optimum oxygenconcentration threshold for storing a chlorophyll containing product orfor storing such a product, the oxygen concentration may initially belower than the optimum threshold, and the threshold found byprogressively increasing the oxygen concentration. In this case, thethreshold may be signified by a transition in which the change in Foand/or the change in Fv/Fm decreases as the oxygen concentration isincreased.

Nitrogen Flush Experiment

FIGS. 32A and 32B show graphs of Fa and minimal fluorescence Fo inresponse to a simulated nitrogen flush accident in which the oxygenlevel in a storage container containing Summerland McIntosh applesremained at very low levels for a period of time. Both FIGS. 32A and 32Bshow that both Fa and Fo of the chlorophyll fluorescence signal emittedby the apples increased as the oxygen level decreased, indicating achange in their health attributable to low oxygen stress. Thus,embodiments of the fluorescence monitoring apparatus and method can beused independently to detect the presence of oxygen levels which wouldbe detrimental to the health of produce which may occur for exampleduring a CA storage nitrogen flush where the oxygen levels fail toreturn to a healthy level.

In other embodiments of the present invention, a plurality offluorescence monitoring devices may be used, each for example comprisinga device as shown in FIG. 1. The devices may be controlled by onecomputer and inter communication between the computer and the devicesmay be made via a hub, connecting the devices to a computer. Differentdevices may be controlled by different computers.

A monitoring device may have any number of individual light sourceelements and light sensors.

In embodiments where the light level is varied, the light level may beactinic, non-actinic or cover a range of non-actinic to actinic levels.

Modifications, alternatives and equivalents to the embodiments describedabove will be apparent to those skilled in the art.

1. A method of detecting the onset of an at least partially reversiblestress condition in chlorophyll-containing matter caused by saidchlorophyll-containing matter being exposed to a stress inducingenvironmental condition, the method comprising: (a) exposing the matterto light to cause chlorophyll in the matter to fluoresce and emit afluorescence signal, (b) detecting the emitted fluorescence signal, (c)measuring the value of a parameter based on the detected fluorescencesignal, (d) monitoring the value of said parameter, (e) detectingchanges in the value of said parameter, (f) providing a threshold valueof a predetermined level of change in said parameter which only ifreached and exceeded signifies the onset of said at least partiallyreversible stress condition in the chlorophyll-containing matter causedby said stress inducing environmental condition, and (g) comparingchanges in the value of said parameter with said threshold value,wherein a determination that the value of said parameter reaches andexceeds said threshold value signifies the onset of said at leastpartially reversible stress condition in the chlorophyll-containingmatter caused by said stress inducing environmental condition.
 2. Themethod of claim 1, comprising making a plurality of measurements of saidparameter, and reducing the exposure of said matter to said lightbetween each said measurement.
 3. The method of claim 1, comprisingmaking a plurality of measurements of said parameter at substantiallythe same level of photon flux to which said matter is exposed.
 4. Themethod of claim 1, wherein the level of photon flux to which said matteris exposed is below that required to stimulate a maximal fluorescencesignal in said chlorophyll-containing matter.
 5. The method of claim 1,wherein said level of photon flux is substantially that required tostimulate a minimal fluorescence signal Fo.
 6. The method of claim 1,wherein said parameter is the intensity of said fluorescence signal. 7.The method of claim 1, comprising measuring the intensity of thefluorescence signal at each of a plurality of different light levels,and deriving said parameter based on said plurality of fluorescencesignal intensity measurements.
 8. The method of claim 7, wherein saidparameter is based on the relationship between a plurality of measuredfluorescence intensities, each measured at a different light level. 9.The method of claim 8, wherein said parameter is a descriptor of saidrelationship.
 10. The method of claim 9, wherein said parameter is afluorescence intensity based on a plurality of measured fluorescenceintensities.
 11. The method of claim 10, wherein said parameter is thefluorescence intensity, Fα, at a level of light exposure of saidchlorophyll-containing matter to zero photon flux.
 12. The method ofclaim 8, comprising fitting a mathematical expression to a plurality ofmeasured fluorescence intensities and wherein said parameter comprises adescriptor of said mathematical expression.
 13. The method of claim 12,wherein said mathematical expression comprises a polynomial regression.14. The method of claim 13, wherein said polynomial regression comprisesa second order polynomial regression.
 15. The method of claim 14,wherein said parameter comprises the value of a constant which qualifiesa term of said polynomial regression.
 16. The method of claim 7, whereineach of said plurality of different light levels to which saidchlorophyll-containing matter is exposed is below that required tostimulate a maximal fluorescence signal in said chlorophyll-containingmatter.
 17. The method of claim 7, wherein said light comprises redlight.
 18. The method of claim 1, wherein the step of exposing thematter to light comprises exposing said matter to a predetermined levelof photon flux by generating a predefined series of light pulses, saidlevel of photon flux being the integrated photon flux of said series ofpulses.
 19. The method of claim 18, wherein the step of exposing saidmatter to light comprises irradiating said matter sequentially withlight at a plurality of different levels of photon flux wherein eachlevel of photon flux is generated by generating a predefined series oflight pulses, each light level being the integrated photon flux of eachseries of pulses.
 20. The method of claim 19, wherein each differentlight level is generated by changing a parameter defining said series ofpulses.
 21. The method of claim 20, wherein the step of changing theintegrated photon flux to which said matter is exposed comprises atleast one of changing the pulse frequency, changing the pulse width,changing the intensity of the pulses and changing the time over whichsaid series of pulses extends.
 22. The method of claim 21, furthercomprising measuring the intensity of the fluorescence signal emitted inresponse to each of a plurality of said pulses within a series.
 23. Themethod of claim 22, further comprising the step of calculating theaverage value of the fluorescence intensity from said plurality ofintensity measurements.
 24. The method of claim 23, further comprisingcalculating the average fluorescence intensity at each of a plurality ofdifferent values of integrated photon flux.
 25. The method of claim 24,comprising the step of deriving said parameter from said plurality ofcalculated average fluorescence intensities.
 26. The method of claim 1,further comprising monitoring said stress inducing environmentalcondition.
 27. The method of claim 26, wherein the step of monitoringsaid environmental condition comprises monitoring a parameter affectingthe health of said chlorophyll-containing matter.
 28. The method ofclaim 27, said method further comprising the step of recording the valueof said parameter affecting the health of said chlorophyll-containingmatter when said change in said parameter based on the detectedfluorescent signal exceeds said predetermined level.
 29. The method ofclaim 1, further comprising changing the level of exposure of saidchlorophyll-containing matter to a condition.
 30. The method of claim29, wherein the step of changing the level of exposure compriseschanging said condition.
 31. The method of claim 30, wherein changingsaid condition comprises changing said condition between a value that isinsufficient to stress said chlorophyll-containing matter and a valuethat is sufficient to stress said chlorophyll-containing matter.
 32. Themethod of claim 1, further comprising controlling the level of exposureof said chlorophyll-containing matter to said environmental conditionbased on the detection of a change in said parameter above saidpredetermined level.
 33. The method of claim 32, wherein saidenvironmental condition comprises at least one of a material absorbed byor taken up by said chlorophyll-containing matter, the concentration ofa gas or liquid in the atmosphere to which said chlorophyll-containingmatter is exposed, temperature, humidity and the pressure to which saidchlorophyll containing matter is exposed.
 34. The method of claim 1,said method further comprising the steps of performing steps (a), (b)and (c) before said matter is exposed to a stress inducing condition,periodically repeating steps (a), (b) and (c) before said matter isexposed to a stress inducing condition, determining a base level of anychange in said parameter based on two or more measurements of saidparameter made before said matter is exposed to a stress inducingcondition, and determining a level above said base level as saidpredetermined level of change.
 35. The method of claim 34, comprisingrepeating step (c) at predetermined successive intervals of time beforesaid matter is exposed to a stress inducing condition.
 36. The method ofclaim 1, wherein said predetermined level is substantially equal to orgreater than about 1%.
 37. The method of claim 1, wherein said change insaid parameter above said predetermined level of change occurs with anincrease in the intensity of the detected fluorescence signal.
 38. Themethod of claim 1, comprising exposing said chlorophyll-containingmatter to an atmosphere containing a predetermined gas, progressivelychanging the level of said predetermined gas to which saidchlorophyll-containing matter is exposed from a level which isinsufficient to induce stress in said chlorophyll-containing matter to alevel which is sufficient to induce stress in saidchlorophyll-containing matter, and said detecting comprises detectingthe onset of said at least partially reversible stress condition causedby exposing said chlorophyll-containing matter to a level of saidpredetermined gas.
 39. The method of claim 38, wherein said gascomprises oxygen and the step of progressively changing comprisesprogressively reducing the level of oxygen to which said chlorophyllcontaining matter is exposed.
 40. The method of claim 38, wherein saidpredetermined gas comprises carbon dioxide, and the step ofprogressively changing comprises progressively increasing the level ofcarbon dioxide to which said chlorophyll-containing matter is exposed.41. The method of claim 1, wherein step (a) comprises exposing saidchlorophyll-containing matter to at least three different levels oflight to cause chlorophyll in the matter to fluoresce and emit afluorescence signal at each different light level, each different lightlevel being below that required to stimulate a maximal fluorescencesignal in said chlorophyll-containing matter, measuring the intensity ofthe fluorescence signal emitted from the chlorophyll-containing matterat each different light level, and step (c) comprises determining fromthe measured intensities, a relationship between the measuredintensities as a function of a parameter indicative of level of light towhich the chlorophyll-containing matter is exposed, and deriving thevalue of said parameter from said relationship.
 42. The method of claim41, wherein said parameter comprises any one of the fluorescenceintensity, fα, at a level of light exposure of said fα to zero photonflux, and the value of a coefficient of a term of a polynomialregression describing the relationship between said measuredintensities.
 43. An apparatus for detecting the onset of an at leastpartially reversible stress condition in chlorophyll-containing matter,comprising: (a) a light source for causing chlorophyll inchlorophyll-containing matter to fluoresce and emit a fluorescencesignal, (b) a detector for detecting the fluorescence signal, (c)measuring means for measuring the value of a parameter based on thedetected fluorescent signal, (d) monitoring means for monitoring changesin the value of said parameter, (e) a device storing a threshold valueof a predetermined level of change in said parameter, which only ifreached and exceeded signifies the onset of said at least partiallyreversible stress condition in said chlorophyll-containing matter causedby exposure of said chlorophyll-containing matter to a stress inducingenvironmental condition, (f) comparing means which compares measuredchanges in the value of said parameter with said threshold value, and(g) detection means adapted to detect an increase in the change of saidparameter above said threshold value.
 44. The apparatus of claim 43,further comprising a controller for controlling the intensity of saidlight source.
 45. The apparatus of claim 44, wherein said controller isarranged to expose said chlorophyll-containing matter to a predeterminedlevel of photon flux successively at predetermined intervals of time andsaid measuring means is arranged to measure said parameter based on thedetected fluorescence signal emitted in response to each successiveexposure to said predetermined level of photon flux.
 46. The apparatusof claim 44, wherein said controller is arranged to successivelyactivate said light source to cause chlorophyll in said matter tofluoresce and after each activation to reduce the intensity of saidlight source.
 47. The apparatus of claim 43, comprising a controllerarranged to control said light source to emit a predetermined integratedphoton flux by causing said light source to emit a predefined series oflight pulses, said integrated photon flux being the integrated photonflux of said series of light pulses.
 48. The apparatus of claim 47,further comprising means for measuring a parameter based on theintensity of said fluorescence signal emitted in response to said seriesof light pulses.
 49. The apparatus of claim 48, wherein said measuringmeans is arranged to measure the intensity of the fluorescent signalemitted in response to each of a plurality of said pulses within saidsenes.
 50. The apparatus of claim 49, further comprising means arrangedto calculate a value of fluorescence intensity based on said pluralityof fluorescence intensities.
 51. The apparatus of claim 50, wherein saidcalculating means is arranged to calculate said fluorescence intensitybased on the average value of the fluorescence intensity from saidplurality of intensity measurements.
 52. The apparatus of claim 51,wherein said controller is arranged to expose said chlorophyll to aplurality of different levels of integrated photon flux within the rangeof about 0.001 to 20 μmol/m²/s.
 53. The apparatus of claim 47, furthercomprising means for measuring the fluorescence intensity after one ormore of said light pulses in a series of light pulses, when theintensity of light from said light source is substantially zero.
 54. Theapparatus of claim 53, comprising means for subtracting the value of afluorescence intensity measured after a light pulse from the value of afluorescence intensity measured during a light pulse.
 55. The apparatusof claim 54, wherein said predetermined level of change is determinedfrom a plurality of measured fluorescence intensities.
 56. The apparatusof claim 43, comprising a controller for exposing saidchlorophyll-containing matter sequentially to a plurality of differentlight levels.
 57. The apparatus of claim 56, wherein said controller isarranged to expose said chlorophyll-containing matter sequentially to atleast three different light levels.
 58. The apparatus of claim 56,comprising measuring means arranged to measure the intensity of thefluorescence signal at each of said plurality of different light levels,and means for deriving said parameter based on said plurality offluorescence signal intensity measurements.
 59. The apparatus of claim58, wherein said parameter is based on the relationship between at leastthree measured fluorescence intensities, each measured at a differentlight level.
 60. The apparatus of claim 59, wherein said parameter is adescriptor of said relationship.
 61. The apparatus of claim 60, whereinsaid parameter is a fluorescence intensity based on a plurality ofmeasured fluorescence intensities.
 62. The apparatus of claim 61,wherein said parameter is the fluorescence intensity, Fα at a level oflight exposure of said chlorophyll-containing matter to zero photonflux.
 63. The apparatus of claim 59, further comprising means forfitting a mathematical expression to a plurality of measuredfluorescence intensities, and wherein said parameter comprises adescriptor of said mathematical expression.
 64. The apparatus of claim63, wherein said mathematical expression compnses a polynomialregression.
 65. The apparatus of claim 64, wherein said polynomialregression comprises a second order polynomial regression.
 66. Theapparatus of claim 64, wherein said parameter comprises the value of aconstant which qualifies a term of said polynomial regression.
 67. Theapparatus of claim 59, wherein each of said at least three differentlight levels to which said chlorophyll-containing matter is exposed isbelow that required to stimulate a maximal fluorescence signal in saidchlorophyll containing matter.
 68. The apparatus of claim 43, comprisinga controller arranged to irradiate said matter sequentially with lightat a plurality of different levels of photon flux, wherein saidcontroller is arranged to generate each level of photon flux bygenerating a predefined series of light pulses, each light level beingthe integrated photon flux of each series of pulses.
 69. The apparatusof claim 68, wherein said controller is arranged to generate eachdifferent light level by changing a parameter defining said series ofpulses.
 70. The apparatus of claim 69, wherein said controller isarranged to change the integrated photon flux to which said matter isexposed by changing at least one of the pulse frequency, the pulsewidth, the intensity of the pulses, and the time over which said seriesof pulses extends.
 71. The apparatus of claim 69, wherein said measuringmeans is arranged to measure the intensity of the fluorescence signalemitted in response to each of a plurality of said pulses within aseries.
 72. The apparatus of claim 71, said apparatus comprising meansfor calculating the average value of the fluorescence intensity fromsaid plurality of intensity measurements.
 73. The apparatus of claim 72,said apparatus further comprising means for calculating the averagefluorescence intensity at each of a plurality of different values ofintegrated of photon flux.
 74. The apparatus of claim 73, comprisingmeans for deriving said parameter from said plurality of calculatedaverage fluorescence intensities.
 75. The apparatus of claim 43, furthercomprising a monitor for monitoring a parameter affecting the health ofsaid chlorophyll-containing matter.
 76. The apparatus of claim 43,further comprising recording means for recording the value of saidparameter affecting the health of said chlorophyll-containing matterwhen said change in said parameter exceeds said predetermined level. 77.The apparatus of claim 43, further comprising means for controlling thevalue of a parameter affecting the health of said chlorophyll-containingmatter.
 78. The apparatus of claim 43, further comprising a controllerfor controlling the value of said parameter affecting the health of saidchlorophyll-containing matter in response to a change in said measuredparameter above said predetermined level.
 79. An apparatus as claimed inclaim 43, further comprising: a controller arranged to expose saidchlorophyll-containing matter sequentially to at least three differentlight levels to cause said chlorophyll-containing matter to fluoresceand emit a fluorescent signal at each light level, said at least threedifferent light levels each being below that required to stimulate amaximal fluorescence signal in said chlorophyll-containing matter, saidmeasuring means being arranged to measure the intensity of thefluorescent signal emitted from said chlorophyll-containing matter ateach of said different light levels, and determining means arranged todetermine a relationship between said measured fluorescence intensitiesas a function of a parameter indicative of light level to which saidchlorophyll-containing matter is exposed, and to provide, as said valueof said parameter based on the detected fluorescent signal, the value ofa parameter based on said determined relationship.
 80. The apparatus ofclaim 79, wherein said parameter is a descriptor of said relationship.81. The apparatus of claim 79, wherein said descriptor comprises any oneof the fluorescence intensity, fα, at a level of light exposure of saidchlorophyll-containing matter to zero photon flux, and the value of acoefficient of a term of a polynomial regression describing therelationship between said measured intensities.
 82. The apparatus ofclaim 43, wherein said light comprises red light.
 83. The apparatus ofclaim 43, wherein said change in said parameter above said predeterminedlevel of change occurs with an increase in the intensity of the detectedfluorescence signal.
 84. A method of detecting the recovery from an atleast partially reversible stress condition in chlorophyll-containingmatter, the method comprising: (a) providing chlorophyll-containingmatter exposed to a stress affecting environmental condition, (b)exposing the matter to light to cause chlorophyll in the matter tofluoresce and emit a fluorescence signal, (c) detecting the emittedfluorescence signal, (d) measuring the value of a parameter based on thedetected fluorescence signal, (e) monitoring the value of saidparameter, (f) detecting changes in the value of said parameter, (g)providing a threshold value of a predetermined level of change in saidparameter which only if reached and exceeded signifies the recovery fromsaid at least partially reversible stress condition in thechlorophyll-containing matter caused by said stress affectingenvironmental condition, and (h) comparing changes in the value of saidparameter with said threshold value, wherein a determination that thevalue of said parameter reaches and exceeds said threshold valuesignifies recovery from said at least partially reversible stresscondition in the chlorophyll-containing matter caused by said stressaffecting environmental condition.
 85. An apparatus for detecting therecovery from an at least partially reversible stress condition inchlorophyll-containing matter comprising: (a) a light source for causingchlorophyll in chlorophyll-containing matter to fluoresce and emit afluorescence signal, (b) a detector for detecting the fluorescencesignal, (c) measuring means for measuring the value of a parameter basedon the detected fluorescence signal, (d) monitoring means for monitoringchanges in the value of said parameter, (e) a device storing a thresholdvalue of a predetermined level of change in said parameter, which onlyif reached and exceeded signifies the recovery from said at leastpartially reversible stress condition in said chlorophyll-containingmatter caused by exposure of said chlorophyll-containing matter to astress affecting environmental condition, (f) comparing means whichcompares changes in the value of said parameter with said thresholdvalue, and (g) detection means adapted to detect an increase in thechange of said parameter above said threshold value.